Multiply-substituted protease variants with altered net charge for use in detergents

ABSTRACT

Novel protease variants derived from the DNA sequences of naturally-occurring or recombinant non-human proteases are disclosed. The variant proteases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding the naturally-occurring or recombinant protease to generate the substitution of a plurality of amino acid residues in the amino acid sequence of a precursor protease. Protease variants are provided that contain substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more negative or less positive compared to a precursor protease and thus the protease variant is more effective in a low detergent concentration system than a precursor protease. Also provided are protease variants containing substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more positive or less negative compared to a precursor protease and thus the protease variant is more effective in a high detergent concentration system than a precursor protease. 
     Protease variants are provided that contain substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more negative or less positive compared to a precursor protease and thus the protease variant is more effective in a medium detergent concentration system than a precursor protease. Also provided are protease variants containing substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more positive or less negative compared to a precursor protease and thus the protease variant is more effective in a medium detergent concentration system than a precursor protease. 
     Further provided is a method of producing a protease variant that is more effective in a low detergent concentration system, medium detergent concentration system and high detergent concentration system than a precursor protease.

RELATED APPLICATIONS

The present application is a continuation-in-part application of U.S. patent application Ser. No. 08/956,323, filed Oct. 23, 1997, U.S. patent application Ser. No. 08/956,564, filed Oct. 23, 1997, and U.S. patent application Ser. No. 08/956,324, filed Oct. 23, 1997, all of which are hereby incorporated herein in their entirety.

BACKGROUND OF THE INVENTION

Serine proteases are a subgroup of carbonyl hydrolases. They comprise a diverse class of enzymes having a wide range of specificities and biological functions. Stroud, R. Sci. Amer., 131:74-88. Despite their functional diversity, the catalytic machinery of serine proteases has been approached by at least two genetically distinct families of enzymes: 1) the subtilisins and 2) the mammalian chymotrypsin-related and homologous bacterial serine proteases (e.g., trypsin and S. gresius trypsin). These two families of serine proteases show remarkably similar mechanisms of catalysis. Kraut, J. (1977), Annu. Rev. Biochem., 46:331-358. Furthermore, although the primary structure is unrelated, the tertiary structure of these two enzyme families bring together a conserved catalytic triad of amino acids consisting of serine, histidine and aspartate.

Subtilisins are serine proteases (approx. MW 27,500) which are secreted in large amounts from a wide variety of Bacillus species and other microorganisms. The protein sequence of subtilisin has been determined from at least nine different species of Bacillus. Markland, F. S., et al. (1983), Hoppe-Seyler's Z. Physiol. Chem., 364:1537-1540. The three-dimensional crystallographic structure of subtilisins from Bacillus amyloliquefaciens, Bacillus licheniformis and several natural variants of B. lentus have been reported. These studies indicate that although subtilisin is genetically unrelated to the mammalian serine proteases, it has a similar active site structure. The x-ray crystal structures of subtilisin containing covalently bound peptide inhibitors (Robertus, J. D., et al. (1972), Biochemistry, 11:2439-2449) or product complexes (Robertus, J. D., et al. (1976), J. Biol. Chem., 251:1097-1103) have also provided information regarding the active site and putative substrate binding cleft of subtilisin. In addition, a large number of kinetic and chemical modification studies have been reported for subtilisin; Svendsen, B. (1976), Carlsberg Res. Commun., 41:237-291; Markland, F. S. Id.) as well as at least one report wherein the side chain of methionine at residue 222 of subtilisin was converted by hydrogen peroxide to methionine-sulfoxide (Stauffer, D. C., et al. (1965), J. Biol. Chem., 244:5333-5338) and extensive site-specific mutagenesis has been carried out (Wells and Estell (1988) TIBS 13:291-297)

A common issue in the development of a protease variant for use in a detergent formulation is the variety of wash conditions including varying detergent formulations that a protease variant might be used in. For example, detergent formulations used in different areas have different concentrations of their relevant components present in the wash water. For example, a European detergent system typically has about 4500-5000 ppm of detergent components in the wash water while a Japanese detergent system typically has approximately 667 ppm of detergent components in the wash water. In North America, particularly the United States, a detergent system typically has about 975 ppm of detergent components present in the wash water. Surprisingly, a method for the rational design of a protease variant for use in a low detergent concentration system, a high detergent concentration system, and/or a medium detergent concentration system as well as for use in all three types of detergent concentration systems has been developed.

SUMMARY OF THE INVENTION

It is an object herein to provide protease variants containing substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more negative or less positive compared to a precursor protease and thus the protease variant is more effective in a low detergent concentration system than a precursor protease. A low detergent concentration system is a wash system that has less than about 800 ppm of detergent components present in the wash water.

It is another object herein to provide protease variants containing substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more positive or less negative compared to a precursor protease and thus the protease variant is more effective in a high detergent concentration system than a precursor protease. A high detergent concentration system is a wash system that has greater than about 2000 ppm of detergent components present in the wash water.

It is another object herein to provide protease variants containing substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more positive or less negative compared to a precursor protease and thus the protease variant is more effective in a medium detergent concentration system than a precursor protease. A medium detergent concentration system is a system that has between about 800 ppm and about 2000 ppm of detergent components present in the wash water.

It is another object herein to provide protease variants containing substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more negative or less positive compared to a precursor protease and thus the protease variant is more effective in a medium detergent concentration system than a precursor protease. A medium detergent concentration system is a wash system that has between about 800 ppm to about 2000 ppm of detergent components present in the wash water.

It is a further object to provide DNA sequences encoding such protease variants, as well as expression vectors containing such variant DNA sequences.

Still further, another object of the invention is to provide host cells transformed with such vectors, as well as host cells which are capable of expressing such DNA to produce protease variants either intracellularly or extracellularly.

There is further provided a cleaning composition comprising a protease variant of the present invention.

Additionally, there is provided an animal feed comprising a protease variant of the present invention.

Also provided is a composition for the treatment of a textile comprising a protease variant of the present invention.

There is further provided a method of producing a protease variant that is more effective in a low, medium and high detergent concentration system than a precursor protease including:

a) substituting an amino acid at one or more residue positions wherein the substitution alters the charge at that position to make the charge more positive or less negative compared to the precursor protease;

b) substituting an amino acid at one or more residue positions wherein the substitution alters the charge at that position to make the charge more negative or less positive compared to the precursor protease;

c) testing the variant to determine its effectiveness in a high, medium and low detergent concentration system compared to the precursor protease; and

d) repeating steps a)-c) as necessary to produce a protease variant that is more effective in a low, medium and high detergent concentration system than a precursor protease wherein steps a) and b) can be done in any order.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B-1 through 1B-3 depict the DNA (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) for Bacillus amyloliquefaciens subtilisin and a partial restriction map of this gene.

FIG. 2 depicts the conserved amino acid residues among subtilisins from Bacillus amyloliquefaciens (BPN)′ and Bacillus lentus (wild-type).

FIGS. 3A and 3B depict the amino acid sequence of four subtilisins. The top line represents the amino acid sequence of subtilisin from Bacillus amyloliquefaciens subtilisin (also sometimes referred to as subtilisin BPN′) (SEQ ID NO:3). The second line depicts the amino acid sequence of subtilisin from Bacillus subtilis (SEQ ID NO:4). The third line depicts the amino acid sequence of subtilisin from B. licheniformis (SEQ ID NO:5). The fourth line depicts the amino acid sequence of subtilisin from Bacillus lentus (also referred to as subtilisin 309 in PCT WO89/06276) (SEQ ID NO:6). The symbol * denotes the absence of specific amino acid residues as compared to subtilisin BPN′.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, certain geographies have certain wash conditions and, as such, use different types of detergents. For example, Japan uses a low detergent concentration system while Europe uses a high detergent concentration system. As discussed previously, the United States uses a medium detergent concentration system. We have found that different protease variants perform optimally in these different detergent formulations. However, as a result of these observations, one would expect that it would be impossible to find a protease that would work well in all three types of detergents. Surprisingly, this is not the case. A method of rationally designing a protease variant to be used in either a low detergent concentration system or a high detergent concentration system or even a medium detergent concentration system as well as one that works in all three detergent concentration systems has been developed.

We have found that in order to produce a protease variant that is more efficacious in a low detergent concentration system, it is necessary to replace positively charged residue(s) either with negatively charged residue(s) or neutral residue(s) and/or neutral residue(s) with negatively charged residue(s). In contrast, we note that in order to produce a protease variant that is more efficacious in a high detergent concentration system, it is necessary to replace negatively charged residue(s) either with positively charged residue(s) or neutral residue(s) and/or neutral residue(s) with positively charged residue(s). Further, we have found that many of the protease variants useful in the low detergent concentration system and/or the high detergent concentration system also are effective in a medium detergent concentration system. By balancing these changes, it is possible to produce a protease variant that works well in low detergent concentration systems, low and medium detergent concentration systems, medium and high detergent concentration systems, high detergent concentration systems, or all three detergent concentration systems.

The electrostatic charge of any ionizable amino acid side chain with an acidic or basic function assumes in aqueous solution is a function of the pH. The acidic residues Glu and Asp, in an equilibrium process, lose a proton by dissociation between pH 3 and 6 thereby acquiring a negative charge. In a similar fashion, His, Lys, and Arg gradually deprotonate between pH 5 and 8, pH 8.5 and 11.5, and pH 11 and 14, respectively, thereby losing a positive charge. The proton of Tyr OH increasingly dissociates between pH 8.5 and 11.5, whereby Tyr acquires a negative charge. The dissociation range for the carboxy terminus is pH 1 to 4, yielding a negative charge, and for the amino terminus it is pH 8 to 11, accompanied by the loss of a positive charge. The dissociation range for amino acid side chains given here are average values for many proteins but they are known to be affected by unusual structural configurations in some proteins.

The cumulative effect of all charges determines whether a protein has a net positive or net negative charge at a given pH. The pH at which positive and negative charges are equally effective and convey an electrostatically neutral state to a protein is called the isoelectric point (pI). A protein will lose or gain charge when the pH is shifted or when an amino acid with an ionizable side chain residue is added or removed. An increase in net positive charge can be achieved either by replacing a residue that at a given pH is negatively charged with an uncharged or a positively charged residue, leading to a formal charge change of +1 and +2, respectively. By replacing an uncharged side chain residue with one that is protonated at the given pH the formal charge change would be +1. Similarly, net negative charge can be increased by replacing positively and uncharged side chains with negatively charged side chains at the pH of observation and gain a formal in crease of negative charge by −1 and −2, respectively.

A low detergent concentration system includes detergents where less than about 800 ppm of detergent components are present in the wash water. Japanese detergents are typically considered low detergent concentration system as they have approximately 667 ppm of detergent components present in the wash water.

A medium detergent concentration includes detergents where between about 800 ppm and about 2000 ppm of detergent components are present in the wash water. North American detergents are generally considered to be medium detergent concentration systems as they have approximately 975 ppm of detergent components present in the wash water. Brazil typically has approximately 1500 ppm of detergent components present in the wash water.

A high detergent concentration system includes detergents where greater than about 2000 ppm of detergent components are present in the wash water. European detergents are generally considered to be high detergent concentration systems as they have approximately 4500-5000 ppm of detergent components in the wash water.

Latin American detergents are generally high suds phosphate builder detergents and the range of detergents used in Latin America can fall in both the medium and high detergent concentrations as they range from 1500 ppm to 6000 ppm of detergent components in the wash water. As mentioned above, Brazil typically has approximately 1500 ppm of detergent components present in the wash water. However, other high suds phosphate builder detergent geographies, not limited to other Latin American countries, may have high detergent concentration systems up to about 6000 ppm of detergent components present in the wash water.

In light of the foregoing, it is evident that concentrations of detergent compositions in typical wash solutions throughout the world varies from less than about 800 ppm of detergent composition (“low detergent concentration geographies”), for example about 667 ppm in Japan, to between about 800 ppm to about 2000 ppm (“medium detergent concentration geographies”), for example about 975 ppm in U.S. and about 1500 ppm in Brazil, to greater than about 2000 ppm (“high detergent concentration geographies”), for example about 4500 ppm to about 5000 ppm in Europe and about 6000 ppm in high suds phosphate builder geographies.

The concentrations of the typical wash solutions are determined empirically. For example, in the U.S., a typical washing machine holds a volume of about 64.4 L of wash solution. Accordingly, in order to obtain a concentration of about 975 ppm of detergent within the wash solution about 62.79 g of detergent composition must be added to the 64.4 L of wash solution. This amount is the typical amount measured into the wash water by the consumer using the measuring cup provided with the detergent.

Proteases are carbonyl hydrolases which generally act to cleave peptide bonds of proteins or peptides. As used herein, “protease” means a naturally-occurring protease or a recombinant protease. Naturally-occurring proteases include α-aminoacylpeptide hydrolase, peptidylamino acid hydrolase, acylamino hydrolase, serine carboxypeptidase, metallocarboxypeptidase, thiol proteinase, carboxylproteinase and metalloproteinase. Serine, metallo, thiol and acid proteases are included, as well as endo and exo-proteases.

The present invention includes protease enzymes which are non-naturally occurring carbonyl hydrolase variants (protease variants) having a different proteolytic activity, stability, substrate specificity, pH profile and/or performance characteristic as compared to the precursor carbonyl hydrolase from which the amino acid sequence of the variant is derived. Specifically, such protease variants have an amino acid sequence not found in nature, which is derived by substitution of a plurality of amino acid residues of a precursor protease with different amino acids. The precursor protease may be a naturally-occurring protease or recombinant protease.

The protease variants useful herein encompass the substitution of any of the nineteen naturally occurring L-amino acids at the designated amino acid residue positions. Such substitutions can be made in any precursor subtilisin (procaryotic, eucaryotic, mammalian, etc.). Throughout this application reference is made to various amino acids by way of common one—and three-letter codes. Such codes are identified in Dale, M. W. (1989), Molecular Genetics of Bacteria, John Wiley & Sons, Ltd., Appendix B.

The protease variants useful herein are preferably derived from a Bacillus subtilisin. More preferably, the protease variants are derived from Bacillus lentus subtilisin and/or subtilisin 309.

Subtilisins are bacterial or fungal proteases which generally act to cleave peptide bonds of proteins or peptides. As used herein, “subtilisin” means a naturally-occurring subtilisin or a recombinant subtilisin. A series of naturally-occurring subtilisins is known to be produced and often secreted by various microbial species. Amino acid sequences of the members of this series are not entirely homologous. However, the subtilisins in this series exhibit the same or similar type of proteolytic activity. This class of serine proteases shares a common amino acid sequence defining a catalytic triad which distinguishes them from the chymotrypsin related class of serine proteases. The subtilisins and chymotrypsin related serine proteases both have a catalytic triad comprising aspartate, histidine and serine. In the subtilisin related proteases the relative order of these amino acids, reading from the amino to carboxy terminus, is aspartate-histidine-serine. In the chymotrypsin related proteases, the relative order, however, is histidine-aspartate-serine. Thus, subtilisin herein refers to a serine protease having the catalytic triad of subtilisin related proteases. Examples include but are not limited to the subtilisins identified in FIG. 3 herein. Generally and for purposes of the present invention, numbering of the amino acids in proteases corresponds to the numbers assigned to the mature Bacillus amyloliquefaciens subtilisin sequence presented in FIG. 1.

“Recombinant subtilisin” or “recombinant protease” refer to a subtilisin or protease in which the DNA sequence encoding the subtilisin or protease is modified to produce a variant (or mutant) DNA sequence which encodes the substitution, deletion or insertion of one or more amino acids in the naturally-occurring amino acid sequence. Suitable methods to produce such modification, and which may be combined with those disclosed herein, include those disclosed in U.S. Pat. No. RE 34,606, U.S. Pat. No. 5,204,015 and U.S. Pat. No. 5,185,258, U.S. Pat. No. 5,700,676, U.S. Pat. No. 5,801,038, and U.S. Pat. No. 5,763,257.

“Non-human subtilisins” and the DNA encoding them may be obtained from many procaryotic and eucaryotic organisms. Suitable examples of procaryotic organisms include gram negative organisms such as E. coli or Pseudomonas and gram positive bacteria such as Micrococcus or Bacillus. Examples of eucaryotic organisms from which subtilisin and their genes may be obtained include yeast such as Saccharomyces cerevisiae, fungi such as Aspergillus sp.

A “protease variant” has an amino acid sequence which is derived from the amino acid sequence of a “precursor protease”. The precursor proteases include naturally-occurring proteases and recombinant proteases. The amino acid sequence of the protease variant is “derived” from the precursor protease amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. Such modification is of the “precursor DNA sequence” which encodes the amino acid sequence of the precursor protease rather than manipulation of the precursor protease enzyme per se. Suitable methods for such manipulation of the precursor DNA sequence include methods disclosed herein, as well as methods known to those skilled in the art (see, for example, EP 0 328299, WO89/06279 and the US patents and applications already referenced herein).

These amino acid position numbers refer to those assigned to the mature Bacillus amyloliquefaciens subtilisin sequence presented in FIG. 1. The invention, however, is not limited to the mutation of this particular subtilisin but extends to precursor proteases containing amino acid residues at positions which are “equivalent” to the particular identified residues in Bacillus amyloliquefaciens subtilisin. In a preferred embodiment of the present invention, the precursor protease is Bacillus lentus subtilisin and the substitutions are made at the equivalent amino acid residue positions in B. lentus corresponding to those listed above.

A residue (amino acid) position of a precursor protease is equivalent to a residue of Bacillus amyloliquefaciens subtilisin if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in Bacillus amyloliquefaciens subtilisin (i.e., having the same or similar functional capacity to combine, react, or interact chemically).

In order to establish homology to primary structure, the amino acid sequence of a precursor protease is directly compared to the Bacillus amyloliquefaciens subtilisin primary sequence and particularly to a set of residues known to be invariant in subtilisins for which sequence is known. For example, FIG. 2 herein shows the conserved residues as between B. amyloliquefaciens subtilisin and B. lentus subtilisin. After aligning the conserved residues, allowing for necessary insertions and deletions in order to maintain alignment (i.e., avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues equivalent to particular amino acids in the primary sequence of Bacillus amyloliquefaciens subtilisin are defined. Alignment of conserved residues preferably should conserve 100% of such residues. However, alignment of greater than 75% or as little as 50% of conserved residues is also adequate to define equivalent residues. Conservation of the catalytic triad, Asp32/His64/Ser221 should be maintained. Siezen et al. (1991) Protein Eng. 4(7):719-737 shows the alignment of a large number of serine proteases. Siezen et al. refer to the grouping as subtilases or subtilisin-like serine proteases.

For example, in FIG. 3, the amino acid sequence of subtilisin from Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis (carlsbergensis) and Bacillus lentus are aligned to provide the maximum amount of homology between amino acid sequences. A comparison of these sequences shows that there are a number of conserved residues contained in each sequence. These conserved residues (as between BPN′ and B. lentus) are identified in FIG. 2.

These conserved residues, thus, may be used to define the corresponding equivalent amino acid residues of Bacillus amyloliquefaciens subtilisin in other subtilisins such as subtilisin from Bacillus lentus (PCT Publication No. WO89/06279 published Jul. 13, 1989), the preferred protease precursor enzyme herein, or the subtilisin referred to as PB92 (EP 0 328 299), which is highly homologous to the preferred Bacillus lentus subtilisin. The amino acid sequences of certain of these subtilisins are aligned in FIGS. 3A and 3B with the sequence of Bacillus amyloliquefaciens subtilisin to produce the maximum homology of conserved residues. As can be seen, there are a number of deletions in the sequence of Bacillus lentus as compared to Bacillus amyloliquefaciens subtilisin. Thus, for example, the equivalent amino acid for Val165 in Bacillus amyloliquefaciens subtilisin in the other subtilisins is isoleucine for B. lentus and B. licheniformis.

“Equivalent residues” may also be defined by determining homology at the level of tertiary structure for a precursor protease whose tertiary structure has been determined by x-ray crystallography. Equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the precursor protease and Bacillus amyloliquefaciens subtilisin (N on N, CA on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the protease in question to the Bacillus amyloliquefaciens subtilisin. The best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available. ${R\quad {factor}} = \frac{{\Sigma_{h}{{{Fo}(h)}}} - {{{Fc}(h)}}}{\Sigma_{h}{{{Fo}(h)}}}$

Equivalent residues which are functionally analogous to a specific residue of Bacillus amyloliquefaciens subtilisin are defined as those amino acids of the precursor protease which may adopt a conformation such that they either alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the Bacillus amyloliquefaciens subtilisin. Further, they are those residues of the precursor protease (for which a tertiary structure has been obtained by x-ray crystallography) which occupy an analogous position to the extent that, although the main chain atoms of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13 nm of the corresponding side chain atoms of Bacillus amyloliquefaciens subtilisin. The coordinates of the three dimensional structure of Bacillus amyloliquefaciens subtilisin are set forth in EPO Publication No. 0 251 446 (equivalent to U.S. Pat. No. 5,182,204, the disclosure of which is incorporated herein by reference) and can be used as outlined above to determine equivalent residues on the level of tertiary structure.

Some of the residues identified for substitution are conserved residues whereas others are not. In the case of residues which are not conserved, the substitution of one or more amino acids is limited to substitutions which produce a variant which has an amino acid sequence that does not correspond to one found in nature. In the case of conserved residues, such substitutions should not result in a naturally-occurring sequence. The protease variants of the present invention include the mature forms of protease variants, as well as the pro- and prepro-forms of such protease variants. The prepro-forms are the preferred construction since this facilitates the expression, secretion and maturation of the protease variants.

“Prosequence” refers to a sequence of amino acids bound to the N-terminal portion of the mature form of a protease which when removed results in the appearance of the “mature” form of the protease. Many proteolytic enzymes are found in nature as translational proenzyme products and, in the absence of post-translational processing, are expressed in this fashion. A preferred prosequence for producing protease variants is the putative prosequence of Bacillus amyloliquefaciens subtilisin, although other protease prosequences may be used.

A “signal sequence” or “presequence” refers to any sequence of amino acids bound to the N-terminal portion of a protease or to the N-terminal portion of a proprotease which may participate in the secretion of the mature or pro forms of the protease. This definition of signal sequence is a functional one, meant to include all those amino acid sequences encoded by the N-terminal portion of the protease gene which participate in the effectuation of the secretion of protease under native conditions. The present invention utilizes such sequences to effect the secretion of the protease variants as defined herein. One possible signal sequence comprises the first seven amino acid residues of the signal sequence from Bacillus subtilis subtilisin fused to the remainder of the signal sequence of the subtilisin from Bacillus lentus (ATCC 21536).

A “prepro” form of a protease variant consists of the mature form of the protease having a prosequence operably linked to the amino terminus of the protease and a “pre” or “signal” sequence operably linked to the amino terminus of the prosequence.

“Expression vector” refers to a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of said DNA in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, “plasmid” and “vector” are sometimes used interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which are, or become, known in the art.

The “host cells” used in the present invention generally are procaryotic or eucaryotic hosts which preferably have been manipulated by the methods disclosed in U.S. Pat. No. RE 34,606 to render them incapable of secreting enzymatically active endoprotease. A preferred host cell for expressing protease is the Bacillus strain BG2036 which is deficient in enzymatically active neutral protease and alkaline protease (subtilisin). The construction of strain BG2036 is described in detail in U.S. Pat. No. 5,264,366. Other host cells for expressing protease include Bacillus subtilis I168 (also described in U.S. Pat. No. RE 34,606 and U.S. Pat. No. 5,264,366, the disclosure of which are incorporated herein by reference), as well as any suitable Bacillus strain such as B. licheniformis, B. lentus, etc.

Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are capable of either replicating vectors encoding the protease variants or expressing the desired protease variant. In the case of vectors which encode the pre- or prepro-form of the protease variant, such variants, when expressed, are typically secreted from the host cell into the host cell medium.

“Operably linked,” when describing the relationship between two DNA regions, simply means that they are functionally related to each other. For example, a presequence is operably linked to a peptide if it functions as a signal sequence, participating in the secretion of the mature form of the protein most probably involving cleavage of the signal sequence. A promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.

The genes encoding the naturally-occurring precursor protease may be obtained in accord with the general methods known to those skilled in the art. The methods generally comprise synthesizing labeled probes having putative sequences encoding regions of the protease of interest, preparing genomic libraries from organisms expressing the protease, and screening the libraries for the gene of interest by hybridization to the probes. Positively hybridizing clones are then mapped and sequenced.

The cloned protease is then used to transform a host cell in order to express the protease. The protease gene is then ligated into a high copy number plasmid. This plasmid replicates in hosts in the sense that it contains the well-known elements necessary for plasmid replication: a promoter operably linked to the gene in question (which may be supplied as the gene's own homologous promoter if it is recognized, i.e., transcribed, by the host), a transcription termination and polyadenylation region (necessary for stability of the mRNA transcribed by the host from the protease gene in certain eucaryotic host cells) which is exogenous or is supplied by the endogenous terminator region of the protease gene and, desirably, a selection gene such as an antibiotic resistance gene that enables continuous cultural maintenance of plasmid-infected host cells by growth in antibiotic-containing media. High copy number plasmids also contain an origin of replication for the host, thereby enabling large numbers of plasmids to be generated in the cytoplasm without chromosomal limitations. However, it is within the scope herein to integrate multiple copies of the protease gene into host genome. This is facilitated by procaryotic and eucaryotic organisms which are particularly susceptible to homologous recombination.

The gene can be a natural B. lentus gene. Alternatively, a synthetic gene encoding a naturally-occurring or mutant precursor protease may be produced. In such an approach, the DNA and/or amino acid sequence of the precursor protease is determined. Multiple, overlapping synthetic single-stranded DNA fragments are thereafter synthesized, which upon hybridization and ligation produce a synthetic DNA encoding the precursor protease. An example of synthetic gene construction is set forth in Example 3 of U.S. Pat. No. 5,204,015, the disclosure of which is incorporated herein by reference.

Once the naturally-occurring or synthetic precursor protease gene has been cloned, a number of modifications are undertaken to enhance the use of the gene beyond synthesis of the naturally-occurring precursor protease. Such modifications include the production of recombinant proteases as disclosed in U.S. Pat. No. RE 34,606 and EPO Publication No. 0 251 446 and the production of protease variants described herein.

The following cassette mutagenesis method may be used to facilitate the construction of the protease variants of the present invention, although other methods may be used. First, the naturally-occurring gene encoding the protease is obtained and sequenced in whole or in part. Then the sequence is scanned for a point at which it is desired to make a mutation (deletion, insertion or substitution) of one or more amino acids in the encoded enzyme. The sequences flanking this point are evaluated for the presence of restriction sites for replacing a short segment of the gene with an oligonucleotide pool which when expressed will encode various mutants. Such restriction sites are preferably unique sites within the protease gene so as to facilitate the replacement of the gene segment. However, any convenient restriction site which is not overly redundant in the protease gene may be used, provided the gene fragments generated by restriction digestion can be reassembled in proper sequence. If restriction sites are not present at locations within a convenient distance from the selected point (from 10 to 15 nucleotides), such sites are generated by substituting nucleotides in the gene in such a fashion that neither the reading frame nor the amino acids encoded are changed in the final construction. Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished by M13 primer extension in accord with generally known methods. The task of locating suitable flanking regions and evaluating the needed changes to arrive at two convenient restriction site sequences is made routine by the redundancy of the genetic code, a restriction enzyme map of the gene and the large number of different restriction enzymes. Note that if a convenient flanking restriction site is available, the above method need be used only in connection with the flanking region which does not contain a site.

Once the naturally-occurring DNA or synthetic DNA is cloned, the restriction sites flanking the positions to be mutated are digested with the cognate restriction enzymes and a plurality of end termini-complementary oligonucleotide cassettes are ligated into the gene. The mutagenesis is simplified by this method because all of the oligonucleotides can be synthesized so as to have the same restriction sites, and no synthetic linkers are necessary to create the restriction sites.

As used herein, proteolytic activity is defined as the rate of hydrolysis of peptide bonds per milligram of active enzyme. Many well known procedures exist for measuring proteolytic activity (K. M. Kalisz, “Microbial Proteinases,” Advances in Biochemical Engineering/Biotechnology, A. Fiechter ed., 1988). In addition to or as an alternative to modified proteolytic activity, the variant enzymes of the present invention may have other modified properties such as K_(m), k_(cat), k_(cat)/K_(m) ratio and/or modified substrate specificity and/or modified pH activity profile. These enzymes can be tailored for the particular substrate which is anticipated to be present, for example, in the preparation of peptides or for hydrolytic processes such as laundry uses.

In one aspect of the invention, the objective is to secure a variant protease having altered proteolytic activity as compared to the precursor protease, since increasing such activity (numerically larger) enables the use of the enzyme to more efficiently act on a target substrate. Also of interest are variant enzymes having altered thermal stability and/or altered substrate specificity as compared to the precursor. In some instances, lower proteolytic activity may be desirable, for example a decrease in proteolytic activity would be useful where the synthetic activity of the proteases is desired (as for synthesizing peptides). One may wish to decrease this proteolytic activity, which is capable of destroying the product of such synthesis. Conversely, in some instances it may be desirable to increase the proteolytic activity of the variant enzyme versus its precursor. Additionally, increases or decreases (alteration) of the stability of the variant, whether alkaline or thermal stability, may be desirable. Increases or decreases in k_(cat), K_(m) or K_(cat)/K_(m) are specific to the substrate used to determine these kinetic parameters.

In another aspect of the invention, it has been found that protease variants containing substitutions of the amino acids atone or more residue positions so that the substitution alters the charge at that position to make the charge more negative or less positive compared to a precursor protease are more effective in a low detergent concentration than a precursor protease.

In a further aspect of the invention, it has been found that protease variants containing substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more positive or less negative compared to a precursor protease are more effective in a high detergent concentration than a precursor protease.

Further, we have found that many of the protease variants useful in the low detergent concentration system and/or the high detergent concentration system also are effective in a medium detergent concentration system.

These substitutions are preferably made in Bacillus lentus (recombinant or native-type) subtilisin, although the substitutions may be made in any Bacillus protease, preferably Bacillus subtilisins.

Based on the screening results obtained with the variant proteases, the noted mutations in Bacillus amyloliquefaciens subtilisin are important to the proteolytic activity, performance and/or stability of these enzymes and the cleaning or wash performance of such variant enzymes.

Many of the protease variants of the invention are useful in formulating various detergent compositions or personal care formulations such as shampoos or lotions. A number of known compounds are suitable surfactants useful in compositions comprising the protease mutants of the invention. These include nonionic, anionic, cationic or zwitterionic detergents, as disclosed in U.S. Pat. No. 4,404,128 to Barry J. Anderson and U.S. Pat. No. 4,261,868 to Jiri Flora, et al. A suitable detergent formulation is that described in Example 7 of U.S. Pat. No. 5,204,015 (previously incorporated by reference). The art is familiar with the different formulations which can be used as cleaning compositions. In addition to typical cleaning compositions, it is readily understood that the protease variants of the present invention may be used for any purpose that native or wild-type proteases are used. Thus, these variants can be used, for example, in bar or liquid soap applications, dishcare formulations, contact lens cleaning solutions or products, peptide hydrolysis, waste treatment, textile applications, as fusion-cleavage enzymes in protein production, etc. The variants of the present invention may comprise enhanced performance in a detergent composition (as compared to the precursor). As used herein, enhanced performance in a detergent is defined as increasing cleaning of certain enzyme sensitive stains such as grass or blood, as determined by usual evaluation after a standard wash cycle.

Proteases of the invention can be formulated into known powdered and liquid detergents having pH between 6.5 and 12.0 at levels of about 0.01 to about 5% (preferably 0.1% to 0.5%) by weight. These detergent cleaning compositions can also include other enzymes such as known proteases, amylases, cellulases, lipases or endoglycosidases, as well as builders and stabilizers.

The addition of proteases of the invention to conventional cleaning compositions does not create any special use limitation. In other words, any temperature and pH suitable for the detergent is also suitable for the present compositions as long as the pH is within the above range, and the temperature is below the described protease's denaturing temperature. In addition, proteases of the invention can be used in a cleaning composition without detergents, again either alone or in combination with builders and stabilizers.

The present invention also relates to cleaning compositions containing the protease variants of the invention. The cleaning compositions may additionally contain additives which are commonly used in cleaning compositions. These can be selected from, but not limited to, bleaches, surfactants, builders, enzymes and bleach catalysts. It would be readily apparent to one of ordinary skill in the art what additives are suitable for inclusion into the compositions. The list provided herein is by no means exhaustive and should be only taken as examples of suitable additives. It will also be readily apparent to one of ordinary skill in the art to only use those additives which are compatible with the enzymes and other components in the composition, for example, surfactant.

When present, the amount of additive present in the cleaning composition is from about 0.01% to about 99.9%, preferably about 1% to about 95%, more preferably about 1% to about 80%.

The variant proteases of the present invention can be included in animal feed such as part of animal feed additives as described in, for example, U.S. Pat. No. 5,612,055; U.S. Pat. No. 5,314,692; and U.S. Pat. No. 5,147,642.

One aspect of the invention is a composition for the treatment of a textile that includes variant proteases of the present invention. The composition can be used to treat for example silk or wool as described in publications such as RD 216,034; EP 134,267; U.S. Pat. No. 4,533,359; and EP 344,259.

The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.

All publications and patents referenced herein are hereby incorporated by reference in their entirety.

EXAMPLE 1

A large number of protease variants were produced and purified using methods well known in the art. All mutations were made in Bacillus lentus GG36 subtilisin.

The protease variants produced were tested for performance in two types of detergent and wash conditions using a microswatch assay described in “An improved method of assaying for a preferred enzyme and/or preferred detergent composition”, U.S. Ser. No. 09/554,992 which claims priority to U.S. Ser. No. 60/068,796 filed Dec. 24, 1997 and published as International Application No. WO 99/34011.

Tables 1-13 list the variant proteases assayed and the results of testing in two different detergents. All values are given as comparison to the first protease shown in the table (i.e., a value of 1.32 indicates an ability to release 132% of the stain as opposed to the 100% of the first variant in the table).

Column A shows the charge difference of a variant. For column B, the detergent was 0.67 g/l filtered Ariel Ultra (Procter & Gamble, Cincinnati, Ohio, USA), in a solution containing 3 grains per gallon mixed Ca²⁺/Mg²⁺ hardness, and 0.3 ppm enzyme was used in each well at 25° C. (low concentration detergent system). For column C, the detergent was 3.38 g/l filtered Ariel Futur (Procter & Gamble, Cincinnati, Ohio, USA), in a solution containing 15 grains per gallon mixed Ca²⁺/Mg²⁺ hardness, and 0.3 ppm enzyme was used in each well at 40° C. (high concentration detergent system).

TABLE 1 A B C N76D S103A V1041 Q109R 1.00 1.00 N76D S103A V1041 Q109R Q245R +1 0.48 1.41

TABLE 2 A B C V68A N76D S103A V1041 G159D Q236H Q245R 1.00 1.00 V68A N76D S103A V1041 G159D N204D Q236H Q245R −1 1.11 0.03

TABLE 3 A B C V68A N76D S103A V1041 1.00 1.00 T22K V68A N76D S103A V1041 +1 0.74 1.85

TABLE 4 A B C N76D S103A V1041 M222S 1.00 1.00 N76D S103A V1041 N173R M222S 0 0.66 1.84 Q12R N76D S103A V1041 M222S Q245R +1 0.41 5.84

TABLE 5 A B C Q12R N76D S103A 1104T S130T M222S Q245R 1.00 1.00 Q12R N76D S103A 1104T S130T M222S Q245R N261D −1 1.79 0.81 Q12R N76D S103A 1104T S130T R170S N185D M222S N243D Q245R −3 2.87 0.02

TABLE 6 A B C V68A N76D S103A V1041 G159D Q236H 1.00 1.00 V68A N76D S103A V1041 G159D Q236H Q245R +1 0.94 6.80 V68A S103A V1041 G159D A232V Q236H Q245R N252K +3 0.44 20.60

TABLE 7 A B C V68A N76D S103A V1041 G159D A232V Q236H Q245R 1.00 1.00 V68A N76D S103A V1041 G159D P210R A232V Q236H Q245R +1 0.44 2.66

TABLE 8 A B C V68A S103A V1041 G159D A232V Q236H Q245R N252K 1.00 1.00 V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K −1 1.96 0.65

TABLE 9 A B C V68A S103A V1041 G159D A232V Q236H Q245R 1.00 1.00 V68A S103A V1041 G159D A232V Q236H K237E Q245R −2 1.27 0.12

TABLE 10 A B C V68A S103A V1041 G159D A232V Q236H Q245R L257V 1.00 1.00 V68A N76D S103A V1041 G159D A232V Q236H Q245R L257V −1 1.56 0.48

TABLE 11 A B C S103A V1041 G159D A232V Q236H Q245R N248D N252K 1.00 1.00 S103A V1041 G159D L217E A232V Q236H Q245R N248D N252K −1 1.90 0.15

TABLE 12 A B C S103A V1041 S101G G159D A232V Q236H Q245R N248D N252K 1.00 1.00 N76D S103A V104I S101G G159D A232V Q236H Q245R N248D N252K −1 1.28 0.39

TABLE 13 A B C N62D S103A V1041 G159D T213R A232V Q236H Q245R N248D N252K 1.00 1.00 N62D S103A V1041 Q109R G159D T213R A232V Q236H Q245R N248D N252K +1 0.40 1.74

EXAMPLE 2

The following protease variants were made and tested as noted in Example 1.

The variants in Table 14 are protease variants which have both types of substitutions: those which alter the charge at a position to make the charge more negative or less positive and those which alter the charge at a position to make the charge more positive or less negative compared to B. lentus GG36 as well as neutral substitutions that do not affect the charge at a given residue position. This produces protease variants that perform better than a standard in both low detergent concentration systems (column A; 0.67 g/l filtered Ariel Ultra (Procter & Gamble, Cincinnati, Ohio, USA), in a solution containing 3 grains per gallon mixed Ca²⁺/Mg²⁺ hardness, and 0.3 ppm enzyme was used in each well at 25° C.) and high detergent concentration systems (column B; 3.38 g/l filtered Ariel Futur (Procter & Gamble, Cincinnati, Ohio, USA), in a solution containing 15 grains per gallon mixed Ca²⁺/Mg²⁺ hardness, and 0.3 ppm enzyme was used in each well at 40° C.).

TABLE 14 A B N76D S103A V104I 1.00 1.00 V68A S103A V104I G159D A232V Q236H Q245R N252K 1.41 1.85 V68A N76D S103A V104I G159D T213R A232V Q236H Q245R T260A 1.30 1.73 V68A S103A V104I G159D A232V Q236H Q245R N248D N252K 2.77 1.20 V68A S103A V104I N140D G159D A232V Q236H Q245R N252K 2.96 1.42 N43K V68A S103A V104I G159D A232V Q236H Q245R 2.05 1.78 N43D V68A S103A V104I G159D A232V Q236H Q245R N252K 2.00 1.34 V68A N76D S103A V104I G159D A215R A232V Q236H Q245R 1.67 1.45 Q12R V68A N76D S103A V104I G159D A232V Q236H Q245R 2.16 1.72 N76D S103A V104I V147I G159D A232V Q236H Q245R N248S K251R 1.35 1.29 V68A N76D S103A V104I G159D A232V Q236H Q245R S256R 2.01 1.72 V68A N76D S103A V104I G159D Q206R A232V Q236H Q245R 2.09 1.62 S103A V104I G159D A232V Q236H Q245R N248D N252K 1.44 1.41 G20R V6BA S103A V104I G159D A232V Q236H Q245R N248D N252K 1.81 1.72 V68A S103A V104I G159D A232V Q236H Q245R N248D N252K L257R 1.51 1.41 V68A S103A V104I A232V Q236H Q245R N248D N252K 1.04 1.50 N76D S103A V104I G159D A232V Q236H Q245R L257V 1.92 1.09

6 1 1497 DNA B. amyloliquefaciens CDS (96)...(1245) 1 ggtctactaa aatattattc catactatac aattaataca cagaataatc tgtctattgg 60 ttattctgca aatgaaaaaa aggagaggat aaaga gtg aga ggc aaa aaa gta 113 Met Arg Gly Lys Lys Val 1 5 tgg atc agt ttg ctg ttt gct tta gcg tta atc ttt acg atg gcg ttc 161 Trp Ile Ser Leu Leu Phe Ala Leu Ala Leu Ile Phe Thr Met Ala Phe 10 15 20 ggc agc aca tcc tct gcc cag gcg gca ggg aaa tca aac ggg gaa aag 209 Gly Ser Thr Ser Ser Ala Gln Ala Ala Gly Lys Ser Asn Gly Glu Lys 25 30 35 aaa tat att gtc ggg ttt aaa cag aca atg agc acg atg agc gcc gct 257 Lys Tyr Ile Val Gly Phe Lys Gln Thr Met Ser Thr Met Ser Ala Ala 40 45 50 aag aag aaa gat gtc att tct gaa aaa ggc ggg aaa gtg caa aag caa 305 Lys Lys Lys Asp Val Ile Ser Glu Lys Gly Gly Lys Val Gln Lys Gln 55 60 65 70 ttc aaa tat gta gac gca gct tca gtc aca tta aac gaa aaa gct gta 353 Phe Lys Tyr Val Asp Ala Ala Ser Val Thr Leu Asn Glu Lys Ala Val 75 80 85 aaa gaa ttg aaa aaa gac ccg agc gtc gct tac gtt gaa gaa gat cac 401 Lys Glu Leu Lys Lys Asp Pro Ser Val Ala Tyr Val Glu Glu Asp His 90 95 100 gta gca cat gcg tac gcg cag tcc gtg cct tac ggc gta tca caa att 449 Val Ala His Ala Tyr Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile 105 110 115 aaa gcc cct gct ctg cac tct caa ggc tac act gga tca aat gtt aaa 497 Lys Ala Pro Ala Leu His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys 120 125 130 gta gcg gtt atc gac agc ggt atc gat tct tct cat cct gat tta aag 545 Val Ala Val Ile Asp Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys 135 140 145 150 gta gca agc gga gcc agc atg gtt cct tct gaa aca aat cct ttc caa 593 Val Ala Ser Gly Ala Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln 155 160 165 gac aac aac tct cac gga act cac gtt gcc ggc aca gtt gcg gct ctt 641 Asp Asn Asn Ser His Gly Thr His Val Ala Gly Thr Val Ala Ala Leu 170 175 180 aat aac tca atc ggt gta tta ggc gtt gcg cca agc gca tca ctt tac 689 Asn Asn Ser Ile Gly Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr 185 190 195 gct gta aaa gtt ctc ggt gct gac ggt tcc ggc caa tac agc tgg atc 737 Ala Val Lys Val Leu Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile 200 205 210 att aac gga atc gag tgg gcg atc gca aac aat atg gac gtt att aac 785 Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn 215 220 225 230 atg agc ctc ggc gga cct tct ggt tct gct gct tta aaa gcg gca gtt 833 Met Ser Leu Gly Gly Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val 235 240 245 gat aaa gcc gtt gca tcc ggc gtc gta gtc gtt gcg gca gcc ggt aac 881 Asp Lys Ala Val Ala Ser Gly Val Val Val Val Ala Ala Ala Gly Asn 250 255 260 gaa ggc act tcc ggc agc tca agc aca gtg ggc tac cct ggt aaa tac 929 Glu Gly Thr Ser Gly Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr 265 270 275 cct tct gtc att gca gta ggc gct gtt gac agc agc aac caa aga gca 977 Pro Ser Val Ile Ala Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala 280 285 290 tct ttc tca agc gta gga cct gag ctt gat gtc atg gca cct ggc gta 1025 Ser Phe Ser Ser Val Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val 295 300 305 310 tct atc caa agc acg ctt cct gga aac aaa tac ggg gcg tac aac ggt 1073 Ser Ile Gln Ser Thr Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly 315 320 325 acg tca atg gca tct ccg cac gtt gcc gga gcg gct gct ttg att ctt 1121 Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu 330 335 340 tct aag cac ccg aac tgg aca aac act caa gtc cgc agc agt tta gaa 1169 Ser Lys His Pro Asn Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu 345 350 355 aac acc act aca aaa ctt ggt gat tct ttg tac tat gga aaa ggg ctg 1217 Asn Thr Thr Thr Lys Leu Gly Asp Ser Leu Tyr Tyr Gly Lys Gly Leu 360 365 370 atc aac gta caa gcg gca gct cag taa a acataaaaaa ccggccttgg 1265 Ile Asn Val Gln Ala Ala Ala Gln 375 380 ccccgccggt tttttattat ttttcttcct ccgcatgttc aatccgctcc ataatcgacg 1325 gatggctccc tctgaaaatt ttaacgagaa acggcgggtt gacccggctc agtcccgtaa 1385 cggccaactc ctgaaacgtc tcaatcgccg cttcccggtt tccggtcagc tcaatgccat 1445 aacggtcggc ggcgttttcc tgataccggg agacggcatt cgtaatcgga tc 1497 2 382 PRT B. amyloliquefaciens 2 Met Arg Gly Lys Lys Val Trp Ile Ser Leu Leu Phe Ala Leu Ala Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Gly Ser Thr Ser Ser Ala Gln Ala Ala Gly 20 25 30 Lys Ser Asn Gly Glu Lys Lys Tyr Ile Val Gly Phe Lys Gln Thr Met 35 40 45 Ser Thr Met Ser Ala Ala Lys Lys Lys Asp Val Ile Ser Glu Lys Gly 50 55 60 Gly Lys Val Gln Lys Gln Phe Lys Tyr Val Asp Ala Ala Ser Val Thr 65 70 75 80 Leu Asn Glu Lys Ala Val Lys Glu Leu Lys Lys Asp Pro Ser Val Ala 85 90 95 Tyr Val Glu Glu Asp His Val Ala His Ala Tyr Ala Gln Ser Val Pro 100 105 110 Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu His Ser Gln Gly Tyr 115 120 125 Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp Ser Gly Ile Asp Ser 130 135 140 Ser His Pro Asp Leu Lys Val Ala Ser Gly Ala Ser Met Val Pro Ser 145 150 155 160 Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His Gly Thr His Val Ala 165 170 175 Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly Val Ala 180 185 190 Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu Gly Ala Asp Gly Ser 195 200 205 Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn 210 215 220 Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala 225 230 235 240 Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala Ser Gly Val Val Val 245 250 255 Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly Ser Ser Ser Thr Val 260 265 270 Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala Val Gly Ala Val Asp 275 280 285 Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val Gly Pro Glu Leu Asp 290 295 300 Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr Leu Pro Gly Asn Lys 305 310 315 320 Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly 325 330 335 Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Trp Thr Asn Thr Gln 340 345 350 Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys Leu Gly Asp Ser Leu 355 360 365 Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala Ala Ala Gln 370 375 380 3 275 PRT B. amyloliquefaciens 3 Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala 35 40 45 Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala 130 135 140 Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly 145 150 155 160 Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala 165 170 175 Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val 180 185 190 Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn 225 230 235 240 Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys 245 250 255 Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 4 275 PRT B. subtilis 4 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 5 274 PRT B. licheniformis 5 Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val 1 5 10 15 Gln Ala Gln Gly Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Gln Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala 35 40 45 Ser Phe Val Ala Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly 50 55 60 Thr His Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val 65 70 75 80 Leu Gly Val Ala Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn 85 90 95 Ser Ser Gly Ser Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp 100 105 110 Ala Thr Thr Asn Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala 115 120 125 Ser Gly Ser Thr Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg 130 135 140 Gly Val Val Val Val Ala Ala Ala Gly Asn Ser Gly Asn Ser Gly Ser 145 150 155 160 Thr Asn Thr Ile Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val 165 170 175 Gly Ala Val Asp Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly 180 185 190 Ala Glu Leu Glu Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr 195 200 205 Pro Thr Asn Thr Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro 210 215 220 His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu 225 230 235 240 Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu 245 250 255 Gly Ser Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala 260 265 270 Ala Gln 6 269 PRT B. lentus 6 Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 1 5 10 15 His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 35 40 45 Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 65 70 75 80 Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125 Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 130 135 140 Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser 145 150 155 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175 Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 180 185 190 Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 195 200 205 Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 225 230 235 240 Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 245 250 255 Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265 

What is claimed:
 1. A method of producing a subtilisin variant that is more effective than a precursor subtilisin in a low detergent concentration system having less than about 800 ppm detergent components present in the wash water, comprising: a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the overall charge of the precursor subtilisin to make a subtilisin variant having a more negative charge or less positive charge compared to the precursor; b) testing the variant in a low detergent system having less than about 800 ppm detergent components present in the wash water by comparing the ability of the precursor and the variant to remove a stain wherein the effectiveness of the precursor is given a value of 1.0 and the variant with improved effectiveness achieves a value greater than 1.0; and c) producing said variant having improved effectiveness.
 2. The method according to claim 1, wherein the variant is derived from a Bacillus subtilisin.
 3. The method according to claim 2, wherein the variant is derived from Bacillus lentus subtilisin.
 4. The method according to claim 1, wherein the detergent composition is formulated into a powdered or liquid detergent having a ph between 6.5 and 12.0.
 5. A method of producing a subtilisin variant that is more effective than a precursor subtilisin in a medium detergent concentration system having between about 800 ppm and about 2000 ppm detergent components in the wash water comprising: substituting an amino acid residue at one or more position in a precursor subtilisin wherein the substitution alters the overall charge of the precursor subtilisin to make a subtilisin variant having a more positive charge or less negative charge compared to the precursor; b) testing the variant in medium detergent system having between about 800 ppm and about 2000 ppm detergent components present in the wash water by comprising the ability of the precursor and the variant to remove a stain wherein the effectiveness of the precursor is given a value of 1.0 and the variant with improved effectiveness achieves a value greater than 1.0; and c) producing said variant having improved effectiveness.
 6. The method according to claim 5, wherein the variant is derived from a Bacillus subtilisin.
 7. The method according to claim 6, wherein the variant is derived from Bacillus lentus subtilisin.
 8. A method of producing a subtilisin variant that is more effective than a precursor subtilisin in a medium detergent concentration system having between about 800 ppm and about 2000 ppm detergent components present in the wash water comprising: a) substituting an amino acid residue at one or more position in a precursor subtilisin wherein the substitution alters the overall charge of the precursor to make a subtilisin variant having a more negative charge or less positive charge compared to the precursor; b) testing the variant in a medium detergent system having between about 800 ppm and about 2000 ppm detergent components present in the wash water by comparing the ability of the precursor and the variant to remove a stain wherein the effectiveness of the precursor is given a value of 1.0 and the variant with improved effectiveness achieves a value greater than 1.0; and c) producing said variant having improved effectiveness.
 9. The method according to claim 8, wherein the variant is derived from a Bacillus subtilisin.
 10. The method according to claim 9, wherein the variant derived from Bacillus lentus is subtilisin.
 11. A method for selecting a subtilisin variant that is more effective than a precursor subtilisin in both a low detergent concentration system and a high detergent concentration system comprising: a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the charge of the position to make the charge more positive or less negative compared to the precursor subtilisin; b) substituting an amino acid residue at one or more positions in the precursor subtilisin wherein the substitution alters the charge of the position to make the charge more negative or less positive compared to the precursor subtilisin; c) obtaining a subtilisin variant of step a) and step b) wherein the overall change in charge compared to the precursor is neutral or positive; d) testing the variant obtained in step c) in both a low detergent concentration system having less than about 800 ppm detergent components present in the wash water and a high detergent concentration system having greater than about 2000 ppm detergent components present in the wash water; and e) selecting the variant having a higher stain release effectiveness than said precursor subtilisin in both the low concentration system and the high concentration system wherein the effectiveness of the precursor is given a value of 1.0 and the variant achieves a value of greater than 1.0.
 12. The method according to claim 11, wherein the overall change in charge of the variant compared to the precursor is neutral.
 13. The method according to claim 11, wherein the overall change in charge of the variant compared to the precursor is positive
 1. 14. The method according to claim 11, wherein step b) is performed before step a).
 15. The method according to claim 11, wherein the variant is derived from a Bacillus subtilisin.
 16. A method for selecting a subtilisin variant that is more effective than a precursor subtilisin in a low detergent concentration system having less than about 800 ppm detergent components present in the wash water, comprising: a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the charge of the position to make the charge more negative or less positive compared to the precursor; b) obtaining a subtilisin variant of step a) wherein the overall change in charge compared to the precursor is negative; c) testing the variant obtained in step b) in a low detergent concentration system having less than about 800 ppm detergent components present in the wash water; and selecting the variant having a higher stain release effectiveness in the low detergent concentration system than said precursor or wherein the effectiveness of the precursor is given a value of 1.0 and the variant achieves a value of greater than 1.0.
 17. The method according to claim 16, in the variant is derived from a Bacillus subtilisin.
 18. A method for selecting a subtilisin variant that is more effective than a precursor subtilisin in a medium detergent concentration system comprising: a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the charge of the position to make the charge more negative or less positive compared to the precursor protease; b) obtaining a subtilisin variant of step a) wherein the overall change in charge compared to the precursor is negative; c) testing the variant obtained in step b) in a medium detergent concentration system having between about 800 ppm and about 200 ppm detergent components present in the wash water; and d) selecting the variant having a higher stain release effectiveness in the medium detergent concentration system than said precursor wherein the effectiveness of the precursor is given a value of 1.0 and the variant achieves a value of greater than 1.0.
 19. The method according to claim 18, wherein the variant is derived from a Bacillus subtilisin.
 20. A method for selecting a subtilisin variant that is more effective than a precursor subtilisin in a medium detergent concentration system, comprising: a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the charge of the position to make the charge more positive or less negative compared to the precursor; b) obtaining a subtilisin protease variant of step a) wherein the overall change in charge compared to the precursor is positive; c) testing the variant obtained in step b) in a medium detergent concentration system having between about 800 ppm to about 2000 ppm detergent components present in the wash water; and d) selecting the variant having a stain release effectiveness in the medium detergent concentration system than said precursor wherein the effectiveness of the precursor is given a value of 1.0 and the variant achieves a value of greater than 1.0.
 21. The method according to claim 20, wherein the variant is derived from Bacillus subtilisin. 